Alternating splicing factor/splicing factor 2 (ASF/SF2), a member of the serine-arginine (SR) protein family, plays important roles both in constitutive and alternative pre-mRNA splicing. Genetic studies show that both the absence and over-expression of ASF/SF2 dramatically change splicing pattern of key genes leading to several diseases including cancer. This suggests the vital roles of ASF/SF2 in cell physiology. Although the precise mode of how ASF/SF2 regulates splicing is not clear, it is thought that its ability to bind exonic enhancer sequences (ESE) through the N-terminal RNA recognition motifs (RRMs) is important. In addition, cycle of phosphorylation and dephosphorylation of the C-terminal region enriched in arginine-serine (RS) dipeptides during splicing is also critical for splicing. We have discovered that the RS domain of ASF/SF2 is modular in terms of phosphorylation. SR protein kinase 1 (SRPK1) processively phosphorylates only the N-terminal part of the RS domain generating hypo-phosphorylated ASF/SF2 (p-ASF/SF2). This partially phosphorylated ASF/SF2 is the substrate of another SR kinase, CLK1 for full or hyper-phosphorylation (pp-ASF/SF2). This proposal will test the hypothesis that the switch between p-ASF/SF2 and pp-ASF/SF2 is dictated by structural modularity of the RS domain, and differential phosphorylation impacts on ESE binding and hence splicing. To test our hypothesis, this proposal focuses on the elucidation of the mechanisms of i) sequential phosphorylation of ASF/SF2 by SRPK1 and CLK1, ii) the role of ASF/SF2 phosphorylation in ESE recognition and iii) the role of RS domain phosphorylation in splicing. We will use x-ray crystallography to study how SRPK1 limits phosphorylation to the N- terminal part of the RS domain and how p-ASF/SF2 phosphorylated by SRPK1 becomes a substrate of CLK1. We will perform biochemistry and structural studies to test how ASF/SF2 recognizes wild type but not oncogenic variants of ESEs. Finally, we will explore the roles of ASF/SF2 phosphorylation in both constitutive and alternative splicing in vitro, and in vivo. PUBLIC HEALTH RELEVANCE: The SR protein family of splicing factors, which regulates alternative and constitutive splicing of all pre-mRNAs by binding to exonic splicing enhancer (ESE) sequences, contain large number of serines that are processively phosphorylated by the SR protein kinase. The focus of this proposal is to understand the mechanism of SR protein, ASF/SF2, phosphorylation by SRPK1 and the role of phosphorylation in the recognition of ESE.